Annexin 5 apc propidium iodide apoptosis detection kit

OMT (Shanghai Jinsui Biotechnology Co., Ltd., China) and TMZ (Sigam-Aldrich, USA) were dissolved in dimethyl sulfoxide (DMSO) and distilled H₂O at a stock concentration of 0.1 M and then further diluted in culture medium to achieve OMT at 10⁻⁷, 10⁻⁶, and 10⁻⁵ M and TMZ at 100 μM (positive control). Additional materials used included 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Company, USA),an Annexin V-APC/propidium iodide (PI) apoptosis detection kit (BD Biosciences, USA), and a Cell Motility HCS Reagent Assay Kit (Thermo Scientific, USA). Mouse anti-human monoclonal antibodies specific for caspase-3, Bcl-2, and Bax were purchased from Cell Signaling Technology (USA), and the secondary antibody was obtained from Santa Cruz Biotechnology (USA).

We utilized the Annexin V‐APC/propidium iodide (PI) Apoptosis Detection Kit (BD Pharmingen) to investigate cell apoptosis following the manufacturer's guidelines. Briefly, cell supernatants were collected 24 h post‐irradiation. The cells were detached using trypsin (without EDTA (ethylenediaminetetraacetic acid)) and combined with the supernatant. After centrifugation, cells were washed thrice with PBS and then resuspended in binding buffer. Annexin V and PI were added as per the manufacturer's instructions, followed by a 5‐min incubation in the dark at room temperature. Subsequently, flow cytometry (Beckman) was employed for cell analysis.